Quantitative analytical assays are important in many fields of medical diagnostics and research where they are used to estimate quantitatively the concentration of analyte particles in samples.
In the prior art there are multiple standard (or “analogue”) assays that use a known correlation between the amplitude of a measured signal (e.g. absorbance of light through a sample cell, electrical conductivity of the sample, time of passage of a sample through a porous bed, intensity of fluorescence, amplitude of force exerted on the sample, etc.) and the concentration of analyte particles in the sample. The estimate E(C) of the concentration C of the analyte particle is then retrieved as a calibrated function ƒ(A) of the amplitude A of the signal.
An example of a reaction that conforms to the above “analogue” requirements is quantitative PCR (or Real-Time PCR) that allows quantification of the measurable signal (i.e. the increase of the fluorescence from a fluorescent DNA stain). Knowing that the amplification of the presence of the analyte particle is geometrical, one can assess the initial number of DNA copies in the sample by analyzing kinematics of the signal measured at the end of every PCR cycle. The Real-Time PCR technique allows to determine the number of copies, or the concentration of the analyte, in a very wide range of concentrations (i.e. dynamic range (Ω)), however, the precision of the measurement of this kind is significantly lower than for competitive quantitative PCR techniques, e.g. digital PCR techniques.
In Real-Time PCR (RT-PCR) a finite number of analyte particles in the inspected volume is sufficient to achieve a measurable signal, and the signal generally increases in time in a reproducible manner, so that it is possible to define a threshold value of the intensity of fluorescence from the inspected volume and measure the interval between the onset of amplification and the instant at which the intensity is equal the threshold value.
Analogue assays, based on RT-PCR technique, have found a wide variety of applications in biochemistry and diagnostics and is used as a ‘golden standard’ technique for concentration assessment. They present a range of advantages.
First of all, it requires a very small sample and no (or very simple) partitioning for assessment and easily determines relative changes of number of analyte particles/concentration of the analyte. Analogue PCR techniques are also relatively quick, as the whole analysis takes only up to one hour, and relatively robust via the detection based on the use of molecular probes.
However, the estimate of concentration of the target nucleic acids in the RT-PCR procedures is always done via referencing to the signal from an external calibrated reference sample containing a known concentration of the target. In practice, due to random and systematic changes in the choice of substrates, analyte particles, sensitivity of the sensor, condition of the apparatus, etc., calibration needs to be performed frequently. Importantly the accuracy of the estimate of concentration obtained via RT-PCR procedure depends on the quality of external calibration and cannot be assessed at the point of measurement.
In the prior art there are also known “digital” assays in which the concentration of analyte particles is established with the use of a statistical calculation on the basis of the number of binary (negative or positive) values of signals recorded from a set of independent partitions of the sample. In the digital assays usually the presence of a single, or a known threshold number of analyte particles, or a threshold concentration of analyte particles in the partition of the sample is amplified to a measurable “positive” signal (“positive” value). As the assays require strong amplification of the presence of the analyte particles, their key application lies in quantitative PCR or ELISA assays.
The development of the concept of the digital assay offered a new paradigm in analytical chemistry. It allows the absolute quantification without calibration of the experimental set-up. Also, it benefits from simplified laboratory routines, i.e. end-point measurement, and relatively plain mathematical tools needed to interpret the experimental results. The digital assays of the prior art are, however, also affected by limitations.
The maximum number of analyte particles M to be determined is directly proportional to the number N of compartments in the assay. In many applications and potential applications of diagnostic quantitation assays it is preferred that the span of the dynamic range (Ω=C+/C−) (with C+ representing the upper limit and C− representing the lower limit of estimated concentration of analyte particles in the assay) is large, for instance is equal to 1 million or more. To reach such a large span of the dynamic range in a standard digital PCR procedure, the sample must be partitioned into proportionally large number of compartments—in the said example—according to the state of knowledge in the field that overlooks unfavourably small precision at very small concentrations, into as many as 200,000 compartments or—actually—as shown in PCT/EP2012/004792, even 600,000 compartments. Partitioning a sample into such a huge number of compartments is—although possible—unfavourable, as such an assay requires specialized, complicated and expensive equipment to be performed. In particular, design of assays that aim to partition, amplify and inspect tens of thousands, or hundreds of thousands or millions of compartments require expensive technologies of micro-fabrication, automation and rapid and sensitive detection from multiple small volumes.
Furthermore, the precision and dynamic range achieved by the digital assays of the prior art cannot be independently tuned narrowing the range of applications and elevating the technical cost of the assays. Thus, it is not possible to obtain high precision (low standard deviation) in a narrow range of concentrations while using a small number N of compartments using digital assays of the prior art.
The prior art provides a solution to increase the span of the dynamic range of a digital assay and for reducing the number of compartments by combining classical digital quantitation assays with different dynamic ranges [Shen, F.; Sun, B.; Kreutz, J. E.; Davydova, E. K.; Du, W.; Reddy P. L.; Joseph, L. J.; Ismagilov, R. F., “Multiplexed Quantification of Nucleic Acids with Large Dynamic Range Using Multivolume Digital RT-PCR on a Rotational SlipChip Tested with HIV and Hepatitis C Viral Load”, J. Am. Chem. Soc., Article ASAP (X 2011)]. The solution relies on a simultaneously performed assay for multiple sets of compartments. The compartments belonging to each set have the same volume. In the cited example, Nz=4 sets are used with Nj=160 compartments each (j=1 to 4), with volumes of compartments equal to vj=1, 5, 25 and 125 nL in each of four sets. The procedure consists in i) dividing the samples into sets and compartments, ii) performing simultaneously signal amplification, iii) counting separately the number Kj of positive values of signals in each of Nz sets and iv) calculating the most probable initial concentration of analyte particles in the sample. The calculation is laborious and demanding, because of the requirement to calculate repeatedly the product
            Π              j        =        1                    N        z              ⁢          {                                                                  [                                                                            N                      j                                        !                                                                                                      K                        j                                            !                                        ⁢                                                                  (                                                                              N                            j                                                    -                                                      K                            j                                                                          )                                            !                                                                      ]                            ⁡                              [                                  1                  -                                      exp                    ⁡                                          (                                                                        -                                                      ν                            j                                                                          ⁢                        C                                            )                                                                      ]                                                    K              j                                ⁡                      [                          exp              ⁡                              (                                                      -                                          ν                      j                                                        ⁢                  C                                )                                      ]                                                N            j                    -                      K            j                              }        ,where the multiplication operator Πj=1Nz denotes the product of Nz terms calculated for each of Nz compartment families, each term comprising the probability of observing Kj positive values of signals from the j-th family. Computation of the result requires an iterative calculation of the said product for each tested, hypothetical value of C within the test dynamic range, until a value C is found for which the above product assumes the maximum. The above procedure must be repeated after each measurement of signals from the sample, which hinders the analysis of the test result, requires a sufficiently fast electronic device or elongates the time needed to obtain the estimate of the number of analyte particles in the sample. The above procedure would be in particular definitely unfavourable in the case of a large number (e.g. ten, or few tens, or hundred or more) of sets of compartments, characterised by that the compartments have the same volume within one set, but different volumes for each two different sets. Furthermore, WO 2012/100198 A2 discloses methods for performing digital measurements with varying volumes and, thus, widening the dynamic range.
Thus, the analogue assays typically provide for a facile method of quantitation on the basis of a single, or small number of measurements, yet they always require external calibration that constitutes an additional cost and hurdle in execution of the method. In contrast thereto, the digital assays typically provide absolute quantitation that does not require calibration. The drawback of the digital assays, however, is that for the requested precision of the estimate of concentration of analyte particles they typically require partitioning the sample into a large set of independent compartments that need to be treated chemically and physically and measured for the signal.
Accordingly, there exists a need for the provision of an improved quantitation method of analyte particles in a sample of predetermined volume, which                allows for a reduced number of compartments and/or        allows for an adjustable precision and/or dynamic range and/or        does not require external calibration.        